Plasmonic Biosensors

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1 Plasmonic Biosensors Lecture 2/2 Andreas B. Dahlin Biotechnical Physics 1 Outline About plasmonic biosensors: What is a surface-based biosensor? Plasmons in nanoparticles and on surfaces. Examples of plasmonics in biotechnology. The connection to binding kinetics to surfaces: We can now measure Γ! Biotechnical Physics 2 1

isolated enzymes, immunosystems, tissues, organelles or whole cells to detect chemical compounds usually by electrical, thermal or optical signals.

analyte (biological or not) Yrecognition element (biological) Y transducer signal 2017-01-25 Biotechnical Physics 3 Biosensors in Everyday Life Not many!

2 What is a Biosensor? One definition can be found in the handbook from the International Union for Pure and Applied Chemistry (IUPAC): A device that uses specific biochemical reactions mediated by isolated enzymes, immunosystems, tissues, organelles or whole cells to detect chemical compounds usually by electrical, thermal or optical signals. The use of recognition elements (or receptors) is sometimes referred to as affinity based biosensing. analyte (biological or not) Yrecognition element (biological) Y transducer signal Biotechnical Physics 3 Biosensors in Everyday Life Not many! New biosensor technologies are more common in research environments. Glucose sensor changed the life of diabetes patients. By some considered to be the only truly successful biosensor and still developing. Quantitative! Pregnancy tests (a lateral flow assay) detect human chorionic gonadotropin from urine. (Also for ovulation.) Qualitative! ~10 nm Au Yao et al. Biosensors and Bioelectronics 2011 Clearblue Biotechnical Physics 4 2

Sensor Terminology Most sensors, not the least biosensors, need to calibrated. In a calibration experiment the response to known doses of the variable of interest is measured.

3 Sensor Terminology Most sensors, not the least biosensors, need to calibrated. In a calibration experiment the response to known doses of the variable of interest is measured. The sensitivity, dynamic range and detection limit are defined from the calibration curve. sensor response (measured) dynamic range slope = sensitivity zero response detection limit environmental variable (controlled) Biotechnical Physics 5 The Challenge of Specificity Most biosensors need to operate label-free to be useful. This means that they work even if the analyte does not carry any artificial label. signal transduction When operating label-free, the biggest problems in biosensor technology is arguably false positive results. When we search for analytes (fish) in biological samples we will always have a lot of other molecules (fishes) present that can interfere with the detection. bait (probe, receptor, recognition element) sample solution target (analyte) Biotechnical Physics 6 3

Surface Sensitive Techniques Detect anything that binds to a surface!

In this course you will learn about two techniques: Surface Plasmon Resonance (SPR) and Quartz Crystal Microbalance (QCM).

com/ 2017-01-25 Biotechnical Physics 7 Know Your Techniques! You will learn the physics behind the techniques.

4 Surface Sensitive Techniques Detect anything that binds to a surface! Several instruments exist, most are based on optical measurements, some mechanical techniques and also a few other. In this course you will learn about two techniques: Surface Plasmon Resonance (SPR) and Quartz Crystal Microbalance (QCM). Biacore (GE Healthcare) Qsense (Biolin Scientific) Biotechnical Physics 7 Know Your Techniques! You will learn the physics behind the techniques. The instruments will not be black boxes to you when you encounter them in the future! Sometimes things go wrong when scientists use machines they know nothing about Rich & Myszka Journal of Molecular Recognition Biotechnical Physics 8 4

Surface-Based Detection The surface must be chemically functionalized such that only the analyte binds. The techniques work in real-time, which gives information about binding kinetics.

5 Surface-Based Detection The surface must be chemically functionalized such that only the analyte binds. The techniques work in real-time, which gives information about binding kinetics. Optical techniques like SPR often enable multiplexing, detection of multiple targets, by imaging mode. sensor surface Y Y Y Y other molecules binding to recognition element Y Y Y Y analyte binding to surface functionalization Y Y Y Y other molecules binding to surface Y Y Y Y recognition element Y inert background analyte sample solution specific interaction Y Y Y Y Biotechnical Physics 9 Biosensor Scenarios In vivo: Inside the living organism. Not for plasmonic biosensors! In situ: Biological sample analyzed in artificial setting. Possible but difficult! In vitro: Artificial setting, bottom-up synthetic biology. Perfect! Biotechnical Physics 10 5

Implications from Binding Kinetics Remember the models for binding kinetics to surfaces! Let the A molecules represent receptors and the B molecules targets (analytes).

Measuring the equilibrium coverage gives only K D from C 0 or vice versa.

6 Implications from Binding Kinetics Remember the models for binding kinetics to surfaces! Let the A molecules represent receptors and the B molecules targets (analytes). A + B AB If the surface is a sensor that gives a measurable response proportional to Γ: Real-time operation needed to determine k on and k off and to confirm equilibrium. Measuring the equilibrium coverage gives only K D from C 0 or vice versa. Diffusion models can estimate performance limits: A molecule can diffuse to the sensor surface without binding to it, but not bind to the surface without first diffusing to it. Flow is often needed to prevent diffusion effects and reach Langmuir behavior. Small sensors give a higher diffusive flux due to edge effects. But the capture efficiency is poor under flow (many molecules will be lost) Biotechnical Physics 11 What is a Plasmon? Physical understanding: Plasmons are collective oscillations in the free electrons of metals. Mathematical understanding: Plasmons are solutions to Maxwell s equations for certain metal-dielectric geometries. For a metal nanoparticle, the polarization enhanced at certain frequencies of light. For noble metals, this occurs for visible light, which gives strong colors. The Lycurgus Cup is the oldest example (year ~400) of this kind of staining. ambient light British Museum light inside cup Freestone et al. Gold Bulletin 2007 Ag, Au, Cu (60%, 30%, 10%) in glass Biotechnical Physics 12 6

Absorption and Scattering The polarizability (α) of the particle determines the absorption and scattering cross sections (σ). The extinction is the sum of absorption and scattering.

7 Absorption and Scattering The polarizability (α) of the particle determines the absorption and scattering cross sections (σ). The extinction is the sum of absorption and scattering. For gold nanoparticles (small and spherical): Blue light is absorbed (true for all gold). Green light is absorbed and scattered (by the plasmon). Red light is transmitted (low extinction). σ σ σ ext sca abs k Im α 4 k 2 6π α σ ext σ sca Here k is the incident wavevector (k = 2π/λ) and α is defined as a volume. The cross sections are areas (shadows)! British Biocell International Biotechnical Physics 13 Electrostatic Approximation By considering the electric field of light as static, the polarizability can be calculated easily for a sphere: α0 3V ε ε εm 2ε m Jackson Classical Electrodynamics Wiley 1999 This is valid for particles that are small compared to the wavelength of light (<50 nm). The symbol ε represents (relative) permittivity at optical frequencies. For simple transparent materials like water, ε is roughly the square of the refractive index (ε = n 2 ). For metals, the permittivity is complex (energy absorption) and dispersive (λ dependent). cross section (σ ext, m 2 ) 8 x R (nm) Au H 2 O geometric cross section areas wavelength (λ 0, nm) Biotechnical Physics 14 7

The Optical Near-Field The absorption and scattering cross sections correspond to the far field properties of the nanoparticles, i.e. they describe what happens to a light wave that passed the particle.

8 The Optical Near-Field The absorption and scattering cross sections correspond to the far field properties of the nanoparticles, i.e. they describe what happens to a light wave that passed the particle. One can also talk about the near-field, i.e. the local electromagnetic field distribution on the nanoscale. This is very important when using the particles as biosensors! The field is strongest at the metal and typically extends a distance approximately equal to the radius of the particle. field enhancement ( E /E 0 ) Electrostatic theory can be used to calculate the near field as well: (y, nm) Au E x, y, z E0 x Re 3 r x 3x xx yy zz 0 r 5 z = 0 λ 0 = 526 nm H 2 O E 0 (x, nm) Biotechnical Physics 15 Surface Plasmons Surface plasmons are similar to ordinary light (electromagnetic plane wave) but confined to the interface between a metal (conductor) and a dielectric (insulator). The surface plasmon propagates along the interface (like a wave on a water surface). Eventually the wave energy has dissipated (normally by heating the metal). The wave must have transverse magnetic polarization. z E field vector in z direction H field vector in y direction wave propagation in x direction H(x,z,t) y 0 z Im(k z ) Re(k x ) dielectric metal E x x distribution x, t E0exp iωtexp ikxx H x z, t H expik x k z ωt of E field, 0 x z Biotechnical Physics 16 y 8

The Dispersion Relation There exists a relation between the momentum and energy of a surface plasmon. In contrast to nanoparticles, surface plasmons exist in a continuum of frequencies!

9 The Dispersion Relation There exists a relation between the momentum and energy of a surface plasmon. In contrast to nanoparticles, surface plasmons exist in a continuum of frequencies! Problem: Momentum is always lacking for the incident light! The dispersion relation does not cross that of photons in the dielectric medium! angular frequency (ω, Hz) x photon in vacuum photon in water H 2 O x 10 7 real part of wavevector (Re(k x ), m -1 ) Biotechnical Physics 17 Au k x c m m Excitation of Surface Plasmons The additional momentum needed can be added in several ways. One is to introduce a periodic grating on the surface. The grating wavevector adds to that of the incident photons. The condition for excitation is: Re j Λ 0 m 0 nm sin 0 m ksin(θ) θ k dielectric Λ Re(k x ) metal One can also use a high refractive index material, a thin metal film and total internal reflection configuration. The resonance condition is then: Re m np sin m dielectric metal k 0 analyze reflection Biotechnical Physics 18 prism θ k 0 n p sin(θ) Re(k x ) 9

10 electric field component (a.u.) Total Internal Reflection When measuring the reflected light one sees a minimum, representing SPR! One can vary either the angle or the wavelength of incident light (spectroscopy at fixed angle) Fresnel coefficient H 2 O Au d = 50 nm n p = 1.7 no full reflection resonance F T F R F A angle of incidence (θ, ) λ nm Biotechnical Physics 19 Im(k Surface Plasmon Near-Field z ) 0 z x Re(k x ) dielectric metal At any time, the surface plasmon has negative and positive poles on the metal surface. The electric field has two components in the xz plane. Magnetic field only in y. distribution of E field The time-averaged field only depends on the distance from the surface due to symmetry. The field extends approximately half of the wavelength of light used to excite the surface plasmon. λ SP k x = i m -1 k z = i m -1 z = 0 2 E z 1 E x H 2 O λ SP /4 Au position in propagation direction (x, μm) Biotechnical Physics 20 10

11 Checklist You do not have to learn any equations for calculating stuff related to the optics, such as how to predict the resonance of a certain nanoparticle etc. However, you need to understand the basic physics. Checklist of some important concepts and the differences between nanoparticle and surface plasmons: Electrostatic approximation. Extinction, absorption and scattering cross sections. Dispersion relation and momentum of incident light. How to excite plasmons by light and spectroscopy methodology. Electromagnetic field extension, difference between near-field and far-field Biotechnical Physics 21 Biosensing with Plasmonics The most common plasmonic biosensor principle is refractometric detection: When a molecule binds to the surface, the refractive index changes. All molecules of interest have a refractive index which is higher than water. The properties of the plasmon are changed because they depend on the refractive index close to the metal. By optical spectroscopy, changes in intensity of light for different wavelengths can then be detected. The resonance shifts in the spectrum. This holds both for surface plasmons (the SPR technique) and nanoparticle plasmons (lab exercise 2) Biotechnical Physics 22 11

Commercial SPR First paper published in 1983. Liedberg et al. Sensors and Actuators 1983 Pharmacia Biosensor started shortly after.

SPR is now the most established biosensor technology for studying biomolecular interactions. But not used for medical diagnostics.

be probed simultaneously using the same sample solution.

12 Commercial SPR First paper published in Liedberg et al. Sensors and Actuators 1983 Pharmacia Biosensor started shortly after. Became Biacore later, which is now part of GE Heathcare. SPR is now the most established biosensor technology for studying biomolecular interactions. But not used for medical diagnostics... The instruments are expensive, but primarily because they contain efficient liquid handling and temperature stabilization etc. SPR can be cheap! Biotechnical Physics 23 SPR Imaging SPR can be operated in imaging mode which enables multiplexing: Several interactions can be probed simultaneously using the same sample solution. One molecule in solution: See how it interacts with different receptors on different spots. Ideal for proteomics and in principle for drug development. Several molecules in solution: Detect the presence of multiple analytes in one sample using different recognition elements. High risk of problems from nonspecific binding. Homola Chemical Society Reviews 2008 ~100 μm spots, ~100 in total Biotechnical Physics 24 12

$Very similar since all are based on refractometric detection! Microvacuum http:/www.owls-sensors.$

13 Other Optical Techniques Plasmons is definitely not the only way to go! Ellipsometry (often for air environment). Optical waveguide lightmode spectroscopy. Dual polarization interferometry. Very similar since all are based on refractometric detection! Microvacuum Biotechnical Physics 25 Importance of Field Extension If the electromagnetic field extends very little, molecules far away will not be detected. If the field extends far, only a part of the detection capability is utilized and the system becomes more sensitive to bulk effects far away from the surface. monolayer gel matrix highest signal nanoparticle Biotechnical Physics 26 13

Miniaturized Nanoparticle Sensors If SPR is so great, why bother with plasmonic biosensors based on nanoparticles?

SPR is hard to make sufficiently small, but through measurements on single nanoparticles one can perhaps reach single

Murray & Barnes Advanced Materials 2008 scattered light in dark field illumination 2017-01-25 Biotechnical Physics 27

14 Miniaturized Nanoparticle Sensors If SPR is so great, why bother with plasmonic biosensors based on nanoparticles? One reason is that people want to resolve individual molecules binding to the surface. SPR is hard to make sufficiently small, but through measurements on single nanoparticles one can perhaps reach single molecule resolution? Murray & Barnes Advanced Materials 2008 scattered light in dark field illumination Biotechnical Physics 27 Resolving Single Molecules Very large proteins adsorbing directly on the surface. Help of complementary techniques can make it slightly better Ament et al. Nano Letters 2012 Zijlstra et al. Nature Nanotechnology 2012 Cool, but is it useful? Biotechnical Physics 28 14

Sandwich Assays Use secondary receptor and signal amplification post binding. Improves detection limit, but excludes real time analysis to get k on and k off!

15 Sandwich Assays Use secondary receptor and signal amplification post binding. Improves detection limit, but excludes real time analysis to get k on and k off! Y second tagged receptor Biotechnical Physics 29 Competitive Assays Measure the reduction in receptor binding to the surface, which contains a receptor for the receptor. Target binding to the receptor in solution blocks binding site for receptor on surface. receptor in solution target Excellent for small molecules and interaction occurs in solution! But again no k on and k off! receptor on surface Y Y Y Y Biotechnical Physics 30 15

$Detection Based on Particle Coupling Instead of detecting changes in refractive index on the metal surface, one can utilize$ In general, the spectral changes are much larger and the sensitivity is better.

In general, the spectral changes are much larger and the sensitivity is better.

link between particles or couples them together.

16 Detection Based on Particle Coupling Instead of detecting changes in refractive index on the metal surface, one can utilize the fact that nanoparticles close to each other will change their plasmon resonances. In general, the spectral changes are much larger and the sensitivity is better. free particles sequence recognition by analyte linked particles However, this requires that the analyte either cleaves the link between particles or couples them together. Y Y Y Y sandwich double recognition Biotechnical Physics 31 Other Uses of Nanoparticles Gold and silver nanoparticles have been used throughout history for improving health. Also, they are great labels since they do not bleach! Wikipedia: Argyria Biotechnical Physics 32 16

17 Reflections & Questions? Biotechnical Physics 33 17

Nanophotonics: principle and application. Khai Q. Le Lecture 11 Optical biosensors

Nanophotonics: principle and application Khai Q. Le Lecture 11 Optical biosensors Outline Biosensors: Introduction Optical Biosensors Label-Free Biosensor: Ringresonator Theory Measurements: Bulk sensing